Triamine chelants, their derivatives, complexes and conjugates

ABSTRACT

A group of functionalized triamine chelants and their derivatives that form complexes with radioactive metal ions are disclosed. The complexes can be covalently attached to a protein or an antibody or antibody fragment and used for therapeutic and/or diagnostic purposes. The chelants are of the formula: ##STR1## wherein n, m, R, R 1 , R 2  and L are defined in the specification.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No.DE-FG02-86ER60400 awarded by the Department of Energy. The Governmenthas certain rights in this invention.

CROSS REFERENCES TO RELATED APPLICATION

The present application is a continuation-in-part application of U.S.application Ser. No. 343,472, filed Apr. 26, 1989, now U.S. Pat. No.5,101,041, issued Mar. 31, 1992.

BACKGROUND OF THE DISCLOSURE

Functionalized chelants, or bifunctional coordinators, are known to becapable of being covalently attached to an antibody having specificityfor cancer or tumor cell epitopes or antigens. Radionuclide complexes ofsuch antibody/chelant conjugates are useful in diagnostic and/ortherapeutic applications as a means of conveying the radionuclide to acancer or tumor cell. See, for example, Meares et al., Anal. Biochem.142, 68-78, (1984); M. W. Brechbiel et al., Inorg. Chem. 25(16),2772-2781 (1986); and Krejcarek et al., Biochem. and Biophys. Res. Comm.77, 581-585 (1977).

Numerous bifunctional chelating agents based on aminocarboxylic acidshave been proposed and prepared. For example the cyclic dianhydride ofDTPA [Hnatowich et al. Science 220, 613-615, (1983); U.S. Pat. No.4,479,930] and mixed carboxycarbonic anhydrides of DTPA [Gansow, U.S.Pat. Nos. 4,454,106 and 4,472,509; Krejcarek et al., Biochem. andBiophys. Res. Comm. 77, 581-585, (1977)] have been reported.

Some chelating agents based on functionalized triamines are known. Forexample, G. H. Searle et al., Aust. J. Chem. 32, 519-36 (1979) teach forthe protection of the terminal nitrogens of linear triamines whichallows the central nitrogen atom to be functionalized with a methylgroup. These compounds were used for the chelation of cobalt ions. Whenthe terminal nitrogen atoms of linear triamines are functionalized withmoieties capable of binding to metal ions using the general methoddisclosed by R. J. Motekaitis et al. [R. J. Motekaitis et al., Inorg.Chem. 23(3), 275-283 (1984)], then a pentadentate chelant is prepared.Additionally the central nitrogen atom is shown to be substituted with abenzyl group. These compounds were also used for the chelation of cobaltions. A. W. Addison et al., Inorg. Chimica Acta 147, 61-64 (1988) and F.Refosco et al., J. Chem. Soc. Dalton Trans. 611-615 (1988) teach thepreparation of a salicylaldehyde Schiff base ligand with a lineartriamine in which the central nitrogen atom is functionalized with amethyl group. These compounds are used for the chelation of iron metalions, and technetium and rhenium metal ions, respectively. E. Chiotelliset al., Nucl. Med. Biol. 15(2), 215-223 (1988) teach the preparation oflinear triamines in which the terminal nitrogen atoms are functionalizedwith alkylthiol moieties. The central nitrogen atom is functionalizedwith a propyl or a cyclohexyl moiety. The compounds were used forcomplexation of ^(99m) Tc.

Bifunctional chelating agents derived from triamines are also known. C.H. Paik et al., J. of Radioanal. Chem. 57(2), 553-564 (1980) teach amethod to prepare functionalized terminal nitrogen atoms of lineartriamines with moieties capable of binding to metal ions and having thecentral nitrogen atom substituted with a benzylamine group forconjugation to protein. These compounds were used for the chelation of¹¹¹ In. Other linear triamine bifunctional chelating agents in which thecentral nitrogen atom is functionalized are disclosed by C. John et al.in J. Nucl. Med. 29, 814-815 (1988) in which the central nitrogen atomis functionalized with a benzylcarboxylic acid group. These compoundswere used for the chelation of ¹⁰⁵ Rh and their conjugation withproteins and antibodies.

SUMMARY OF THE INVENTION

The invention includes the design and synthesis of novel bifunctionalchelants, each containing a chelating functionality, and a chemicallyreactive group for covalent attachment to biomolecules. Also formingpart of the invention are methods for preparing various BFC-metalcomplexes and the linking of the complexes to antibody to prepareradionuclide labeled antibody and/or fragments suitable for diagnosticand/or therapeutic applications.

The present invention is directed to novel bifunctional chelating agents(BFC) having a triamine functionality and derivatives thereof. The BFC'sform complexes with "radioactive" metal ions. Preferred radioactivemetal ions include ^(99m) Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ⁵⁷ Co, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁹⁷Ru, ¹¹¹ In, ^(113m) In, ⁶⁷ Ga, and ⁶⁸ Ga. The complexes so formed can beattached (covalently bonded) to an antibody or fragment thereof to formconjugates and used for therapeutic and/or diagnostic purposes. Thecomplexes and/or conjugates can be formulated for in vivo or in vitrouses. A preferred use of the formulated conjugates is the treatment ofcancer in animals, especially humans.

More specifically, the present invention is directed to a compound ofthe formula: ##STR2## wherein:

R represents independently hydrogen, C₁ -C₃ alkyl, or benzyl;

R¹ represents --CH₂ C(CH₃)₂ SH, --(CH₂)₂ NH₂, --(CH₂)₂ SH, ##STR3##

Q represents hydrogen, C₁ -C₃ alkyl or phenyl;

R² represents hydrogen, --CH₂ CO₂ H, --CH₂ CH₂ CO₂ H, or --(CH₂)₂ NH₂ ;

m and n are independently 2, 3, or 4;

L is a linker/spacer group covalently bonded to, and replaces onehydrogen atom of the nitrogen atom to which it is joined, saidlinker/spacer group being represented by the formula ##STR4## wherein:

s is an integer of 0 or 1;

t is an integer of 0 to 20 inclusive;

R³ is an electrophilic or nucleophilic moiety which allows for covalentattachment to an antibody or fragment thereof, or a synthetic linkerwhich can be attached to an antibody or fragment thereof; and

Cyc represents a cyclic aliphatic moiety, aromatic moiety, aliphaticheterocyclic moiety, or aromatic heterocyclic moiety, each of saidmoieties optionally substituted with one or more groups which do notinterfere with binding to an antibody or antibody fragment; or

a pharmaceutically acceptable salt thereof.

Preferred features of the compounds of formula (I) are those where: R ishydrogen; R² is hydrogen; m and n are independently 2 or 3; and L is acompound of the formula: ##STR5## wherein:

Y is selected from the group consisting of nitro, amino, isothiocyanato,semicarbazido, thiosemicarbazido, carboxyl, bromoacetamido andmaleimido;

q is 1, 2, or 3; or

a pharmaceutically acceptable salt thereof.

When a conjugate of the present invention is desired Y must be otherthan nitro.

Preferred Compounds of formula (I) are those where m and n are 2 or 3,R² is hydrogen, R¹ is ##STR6## L is formula (A) where Y is amino orisothiocyanato, or a pharmaceutically acceptable salt thereof.

The present invention is also directed to radioactive metal ioncomplexes which have as their ligand the compounds of formula (I),especially radioactive metal ion complexes comprising ^(99m) Tc, ¹⁰⁵ Rh,¹⁰⁹ Pd, ⁵⁷ Co, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁹⁷ Ru, ¹¹¹ In, ^(113m) In, ⁶⁷ Ga, and ⁶⁸Ga, with the with the proviso that when R² is --CH₂ CO₂ H or --CH₂ CH₂CO₂ H, then the radioactive metal ion is selected from the groupconsisting of ¹⁸⁶ Re, ¹⁸⁸ Re, ^(99m) Tc, ¹¹¹ In, ^(113m) In, ⁶⁷ Ga, and⁶⁸ Ga.

Additionally, the present invention concerns conjugates which are formedwith the aforementioned complexes and antibody or antibody fragments.

In addition, the present invention also includes formulations having theconjugates of the invention and a pharmaceutically acceptable carrier,especially formulations where the pharmaceutically acceptable carrier isa liquid. The invention also includes a method for the diagnosis ortreatment of a disease state, especially cancer, in a mammal whichcomprises administering to the mammal an effective amount of theformulation.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following indicated terms have these meanings: withrespect to the definition of Y, "electrophilic" moieties include, butare not limited to, isothiocyanate, bromoacetamide, maleimide,imidoester, thiophthalimide, N-hydroxysuccinimyl ester, pyridyldisulfide, and phenyl azide; suitable "nucleophilic" moieties include,but are not limited to, carboxyl, amino, acyl hydrazide, semicarbazide,and thiosemicarbazide; "synthetic linkers" include any synthetic organicor inorganic linkers which are capable of being covalently attached toan antibody or antibody fragment, preferred synthetic linkers arebiodegradable synthetic linkers which are stable in the serum of apatient but which have a potential for enzymatic cleavage within anorgan of clearance for the radioisotope, for example biodegradablepeptides or peptide containing groups. Of the electrophilic moietiesisothiocyanate is preferred and of the nucleophilic moieties amino,carboxyl, semicarbazide and thiosemicarbazide are preferred. It isdesirable that the nature and/or position of Y be such that it does notappreciably interfere with the chelation reaction.

As used herein, the term "mammal" means animals that nourish their youngwith milk secreted by mammary glands, preferably warm blooded mammals,more preferably humans. "Antibody" refers to any polyclonal, monoclonal,chimeric antibody or heteroantibody, preferably a monoclonal antibody;"antibody fragment" includes Fab fragments and F(ab')₂ fragments, andany portion of an antibody having specificity toward a desired epitopeor epitopes. When using the term "metal chelate/antibody conjugate" or"conjugate", the "antibody" portion is meant to include whole antibodiesand/or antibody fragments, including semisynthetic or geneticallyengineered variants thereof.

As used herein, "complex" refers to a compound of the invention, e.g.formula (I), complexed with a radioactive metal ion, wherein at leastone metal atom is chelated or sequestered; "radioactive metal ionchelate/antibody conjugate" or "radioactive metal ion conjugate" refersto a radioactive metal ion conjugate that is covalently attached to anantibody or antibody fragment; "radioactive" when used in conjunctionwith the word "metal ion" refers to one or more isotopes of the elementsthat emit particles and/or photons, such as ^(99m) Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd,⁵⁷ Co, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁹⁷ Ru, ¹¹¹ In, ^(113m) In, ⁶⁷ Ga, and ⁶⁸ Ga; theterms "bifunctional coordinator", "bifunctional chelating agent" and"functionalized chelant" are used interchangeably and refer to compoundsthat have a chelant moiety capable of chelating a metal ion and alinker/spacer moiety covalently bonded to the chelant moiety that iscapable of serving as a means to covalently attach to an antibody orantibody fragment.

As used herein, "pharmaceutically acceptable salt" means any salt of acompound of formula (I) which is sufficiently non-toxic to be useful intherapy or diagnosis of mammals. Thus, the salts are useful inaccordance with this invention. Representative of those salts, which areformed by standard reactions, from both organic and inorganic sourcesinclude, for example, sulfuric, hydrochloric, phosphoric, acetic,succinic, citric, lactic, maleic, fumaric, palmitic, cholic, palmoic,mucic, glutamic, d-camphoric, glutaric, glycolic, phthalic, tartaric,formic, lauric, steric, salicylic, methanesulfonic, benzenesulfonic,sorbic, picric, benzoic, cinnamic acids and other suitable acids. Alsoincluded are salts formed by standard reactions from both organic andinorganic sources such as ammonium, alkali metal ions, alkaline earthmetal ions, and other similar ions. Particularly preferred are the saltsof the compounds of formula (I) where the salt is potassium, sodium,ammonium, H⁺ or mixtures thereof.

The bifunctional chelating agents described herein can be used tochelate or sequester the radioactive metal ions, so as to form metal ionchelates (also referred to herein as "complexes"). The complexes,because of the presence of the functionalizing moiety [represented by"Y" in formula (I)], can be attached to functionalized supports, such asfunctionalized polymeric supports, or preferably covalently attached toproteins or more specifically to antibodies or antibody fragments. Thusthe complexes described herein complexed with radioactive metal ions maybe covalently attached to a protein or an antibody or antibody fragmentand are referred to herein as "conjugates". For example, human serumalbumin (HSA), purchased from Sigma, was used to form conjugates with aprotein.

The antibodies or antibody fragments which may be used in the conjugatesdescribed herein can be prepared by techniques well known in the art.Highly specific monoclonal antibodies can be produced by hybridizationtechniques well known in the art, see for example, Kohler and Milstein[Nature 256, 495-497 (1975); and Eur. J. Immunol. 6, 511-519 (1976)].Such antibodies normally have a highly specific reactivity. In theradioactive metal ion conjugates, antibodies directed against anydesired antigen or hapten may be used. Preferably the antibodies whichare used in the radioactive metal ion conjugates are monoclonalantibodies, or fragments thereof having high specificity for a desiredepitope(s). Antibodies used in the present invention may be directedagainst, for example, tumors, bacteria, fungi, viruses, parasites,mycoplasma, differentiation and other cell membrane antigens, pathogensurface antigens, toxins, enzymes, allergens, drugs and any biologicallyactive molecules. Some examples of antibodies or antibody fragments areB72.3 and IgG. [The hybridoma cell line B72.3 is deposited in theAmerican Type Culture Collection (ATCC) having the accession number HB8108. The IgG was purchased from Sigma.] A more complete list ofantigens can be found in U.S. Pat. No. 4,193,983, which is incorporatedherein by reference. The radioactive metal ion conjugates of the presentinvention are particularly preferred for the diagnosis and treatment ofvarious cancers.

The conjugates of this invention, and in some instances the complexes,are employed as a formulation. The formulation comprises a compound offormula (I) with the antibody and/or radioactive metal ion and aphysiologically acceptable carrier, excipient or vehicle therefor. Thus,the formulation may consist of a physiologically acceptable carrier witha complex (metal ion+ligand), conjugate (metal ion+ligand+antibody). Themethods for preparing such formulations are well known. The formulationmay be in the form of a suspension, injectable solution or othersuitable formulation. Physiologically acceptable suspending media, withor without adjuvants, may be used.

The formulations of the present invention are in the solid or liquidform containing the active radionuclide complexed with the ligand. Theseformulations may be in kit form such that the two components (i.e.ligand and metal, complex and antibody) are mixed at the appropriatetime prior to use. Whether premixed or as a kit, the formulationsusually require a pharmaceutically acceptable carrier.

Injectable compositions of the present invention may be either insuspension or solution form. In the preparation of suitable formulationsit will be recognized that, in general, the water solubility of the saltis greater than the free base. In solution form the complex (or whendesired the separate components) is dissolved in a physiologicallyacceptable carrier. Such carriers comprise a suitable solvent,preservatives such as benzyl alcohol, if needed, and buffers. Usefulsolvents include, for example, water, aqueous alcohols, glycols, andphosphonate or carbonate esters. Such aqueous solutions contain no morethan 50 percent of the organic solvent by volume.

Injectable suspensions are compositions of the present inventionrequiring a liquid suspending medium, with or without adjuvants, as acarrier. The suspending medium can be, for example, aqueouspolyvinylpyrrolidone, inert oils such as vegetable oils or highlyrefined mineral oils, or aqueous carboxymethylcellulose. Suitablephysiologically acceptable adjuvants, if necessary to keep the complexin suspension, may be chosen from among thickners such ascarboxymethylcellulose, polyvinylpryrrolidone, gelatin, and thealginates. Many surfactants are also useful as suspending agents, forexample, lecithin, alkylphenol, polyethylene oxide adducts,napthalenesulfonates, alkylbenzenesulfonates, and the polyoxyethylenesorbitan esters.

Many substances which effect the hydrophobicity, density, and surfacetension of the liquid suspension medium can assist in making injectablesuspensions in individual cases. For example, silicone antifoames,sorbitol, and sugars are all useful suspending agents.

An "effective amount" of the formulation is used for therapy. The dosewill vary depending on the disease being treated. Although in vitrodiagnostics can be performed with the formulations of this invention, invivo diagnostics are also contemplated using formulations of thisinvention. The conjugates and formulations of this invention can also beused in radioimmunoguided surgery (RIGS); however, the metals whichcould be used for this purpose are ^(99m) Tc, ¹¹¹ In, ^(113m) In, ⁶⁷ Gaand ⁶⁸ Ga.

The present invention provides chelants, complexes, and antibodyconjugates some of which are more stable, and/or have improvedbiodistribution, and/or have more rapid clearance from the body, thanthose known in the art.

DETAILED DESCRIPTION OF THE PROCESS

All of the starting materials required for preparing the compounds ofthis invention are either available from commercial sources or can bemade from a known literature reference description.

The electrophilic moiety ("Y" in the formula) can also be prepared bymethods known to the art. Such methods may be found in Acc. Chem. Res.17, 202-209 (1984).

General methods of preparation for the compounds of formula (I) are wellknown in the art. Some of the various methods are shown in the followingreaction schemes.

In the following Scheme I, the terminal amines of a symmetrical orunsymmetrical triamine are protected by the addition of phthalicanhydride using a modification of the procedure of G. H. Searle, et al.,Aust. J. Chem. 32, 519-36 (1979). The central amine is then alkylatedwith the L moiety. ##STR7##

In Scheme II, the protecting phthalic moieties of the product fromScheme I are removed by hydrolysis with hydrochloric acid. Thep-nitrobenzyl group is then reduced by conventional methods, i.e. Pd/Cwith H₂. The compounds of formula (I) where R¹ is an oxime are preparedby the addition of 3-chloro-3-methyl-2-butanone oxime. ##STR8##

In Scheme III, the protecting phthalic moieties are removed byhydrolysis with hydrochloric acid. The Schiff bases are then prepared byreaction with the appropriate aldehyde. Reduction of the Schiff basesproceeds with sodium borohydride. The nitro group is then reduced to theamine using Pd/C with H₂. ##STR9##

In Scheme IV, the protecting phthalic moieties are removed by hydrolysiswith hydrochloric acid. The compounds of formula (I) where Y is nitroand the R¹ groups are the correspondingly protected aminoamide orthioamide are then formed by reaction with the corresponding alkyl aminoester (e.g. methyl 2-(tBOC)-aminoacetate) or alkylthio ester (e.g.methyl 2-mercaptoacetate). When the tertiary butylcarbamate (tBOC) ispresent, it is cleaved by trifluoroacetic acid.

For compounds having either the unprotected aminoamide or thioamidemoiety, the nitro group is then reduced to the amine using Pd/C with H₂.

Additionally, for compounds where Y is nitro having either theunprotected amino amide or thioamide moieties, reduction with boraneleads to the corresponding alkyl amines or alkyl sulfhydryls. Subsequentreduction of the nitro group results in the corresponding amines.##STR10##

In Scheme V, to prepare the compounds of formula (I) where R² is otherthan hydrogen, the corresponding compounds of formula (I) where R² ishydrogen are reacted in one of the following manners.

The compounds of formula (I) wherein R² is alkyl amine are prepared by amodification of the Strecker reaction (aldehyde reaction with hydrogencyanide, followed by reduction with lithium aluminum hydride).

The compounds of formula (I) wherein R² is a carboxylic acid areprepared by the addition of bromocarboxylic acid. See for example, J. F.Desreux, Inorg. Chem. 19, 1319-1324 (1980). ##STR11##

In Scheme VI, to prepare the compound of formula (I) where R¹ and R² are--CH₂ --C(CH₃)₂ SH, the corresponding compounds of formula (I) whereinR¹ and R² are both hydrogen is reacted with a bisdithiaaldehyde, such asCHO--(CH₃)₂ --S--S--(CH₃)₂ --CHO, after which reaction R¹ and R² areboth ═C--C(CH₃)₂ --SH, which moieties are reduced with a suitablereducing agent, such as LiAlH₄, to provide the desired compound offormula (I). ##STR12##

Radionuclides can be produced in several ways. For example, severalpreparations are detailed in an article by D. E. Troutner, Nucl. Med.Biol., 14(3), 171-176 (1987). The method of obtaining the nuclidesemployed in the present invention is not critical thereto.

The complexes of the present invention are generally prepared by mixingthe ligand with the desired radioactive metal ion. However, when theradioactive metal ion is ¹⁸⁶ Re, ¹⁸⁸ Re or ^(99m) Tc, then a reducingagent must be added such as tin tartarate. When the radioactive metalion is ⁹⁷ Ru or ¹⁰⁵ Rh, then the mixture must be heated.

Attachment of radionuclide to antibody can be carried out by conjugationof the antibody to preformed (ambient or elevated temperature)metal--BFC complex. See European published application 0296522,published Dec. 28, 1988]. The conjugates of the present invention areprepared by first activating the complex with thiophosgene whichconverts the amine, where Y is NH₂, to the corresponding isothiocyanate.The desired protein or antibody or antibody fragment, in presence ofbuffer, is added to the isothiocyanate compound. A covalent bond isformed (thiourea) by the reaction of the isothiocyanate with thealkylamine of a lysine group.

The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of the useof the invention.

In the following examples, the following terms and conditions were usedunless otherwise specified:

Room temperature means about 25° C.;

Bq means a Becquerel unit for measuring radioactivity. 1 Curie=3.7×10¹⁰Bq;

0.9% Saline solution was prepared by dissolving 9 g of NaCl in 1 L ofdoubly distilled water;

¹⁰⁵ Rh was prepared at the University of Missouri Research Reactor(MURR) and supplied as Na₃ (RhCl₆) or Na₂ (RhCl₅.H₂ O) in approximatelypH 4 HCl solution;

Rh carrier solutions were prepared by dissolving RhCl₃.3H₂ O in saline;

^(99m) Tc was eluted from a Mallinckrodt technetium generator anddiluted to 2 mCi/mL using 0.9% saline solution;

Human immunoglobulin (I-4506) was purchased from Sigma Chemical Company;

Sephadex™ G 75 was purchased from Sigma Chemical Company;

Anti-human IgG (γ chain specific), Agarose (A-6656) was purchased fromSigma Chemical Company;

Thiophosgene was purchased from Aldrich Chemical Company;

RhCl₃.3H₂ O was purchased from Aldrich Chemical Company;

Flexible silica gel plates 7.5×2.5 cm, coating thickness 250 μm werepurchased from J. T. Baker Chemical Company;

Beckman series 320046 paper (30×2 cm) was used for paper electrophoresisand Gelman Science solvent saturation pads cut into 1.5×13.7 cm wereused for paper chromatography;

¹ H and ¹³ C NMR spectra were recorded on a JEOL FX-90Q spectrometer,300 MHz with δ reported in ppm relative to TMS as an internal standard;and

Radioactivity was measured on a standard 5×5 cm NaI (T1) wellscintillation counter.

PREPARATION OF STARTING MATERIALS EXAMPLE A

Method A: Preparation of4-(4-nitrobenzyl)-1,7-diphthaloyldiethylenetriamine

To a round bottom flask was added 100 mL of absolute ethanol and 1.6 g(28 mmol) of potassium hydroxide. The resulting mixture was heated untilthe potassium hydroxide dissolved.

Ten g (28 mmol) of N,N'-(iminodiethylene)bisphthalimide [prepared by theprocedure of G. H. Searle et al. Aust. J. Chem. 32, 519-36 (1979)(seepage 531)] was added to the solution and then the reaction mixture wasrefluxed for 2.5 hours. To the mixture was added 5.95 g (28 mmol) ofp-nitrobenzylbromide and the mixture was again refluxed for 16 hours.The solution was filtered hot and upon cooling the filtrate, the productcrystallized, and was filtered to yield 11.0 g of4-(4-nitrobenzyl)-1,7-diphthaloyldiethylenetriamine, mp 128°-130° C. Theproduct was recrystallized from absolute ethanol and furthercharacterized by: ¹ H NMR (CDCl₃) δ 2.6-2.9 (t, 4H), 3.6-3.9 (m, 6H),7.1-7.6 (m, 12H).

Method B: Preparation of4-(4-nitrophenyl)-1,7-diphthaloyldiethylenetriamine

To a round bottom flask was added 100 mL of absolute ethanol and 1.6 g(28 mmol) of potassium hydroxide. The resulting mixture was heated untilthe base dissolved. N,N'-(iminodiethylene)bisphthalimide, 10 g (28 mmol)[prepared by the procedure of Martell et al., Inorg. Chem. 18(11),2982-2986 (1979)] was added to the solution and the reaction mixturerefluxed for 2.5 hours. To the mixture was added 5.95 g (28 mmol) ofp-nitrobenzyl bromide and the mixture was again refluxed for 16 hours.The solution was filtered hot and upon cooling the filtrate, the productcrystalized and was filtered to yield 11 g of4-(4-nitrobenzyl)-1,7-diphthaloyldiethylenetriamine, mp 130° C. Theproduct was reerystalized from absolute ethanol and furthercharacterized by: ¹ H NMR (CDCl₃) δ 2.6-2.9 (t, 4H), 3.6-3.9 (m, 6H),7.1-7.6 (m, 12H).

EXAMPLE B

Preparation of 4-(p-nitrobenzyl)diethylenetriamine

To a round bottom flask was added 3.0 g of4-(p-nitrobenzyl)-1,7-diphthaloyldiethylenetriamine (prepared in ExampleA) and 60 mL of 6M hydrochloric acid. The mixture was refluxed for about16 hours. A clear solution was obtained, which upon cooling produced awhite solid. The solid was filtered and the filtrate evaporated on arotary evaporator to give as a white solid 1.9 g (91%) of4-(p-nitrobenzyl)diethylenetriamine as the hydrochloride salt. This saltwas neutralized with sodium hydroxide solution and extracted intochloroform (3 times with 30 mL portions). The organic layers werecombined and evaporated to yield as an oil,4-(p-nitrobenzyl)diethylenetriamine. ¹ H NMR (CDCl₃) δ 1.65 (s, 4H),2.4-2.9 (m, 8H), 3.7 (s, 2H), 7.3-8.3 (m, 4H).

EXAMPLE C

Preparation of bis(salicylidine)-4-(p-nitrobenzyl)diethylenetriamine.

In 50 mL of ethanol in a round bottom flask was added 700 mg (2.4 mmol)of 4-(p-nitrobenzyl)diethylenetriamine (prepared in Example B) and 720mg (5.9 mmol) of salicylaldehyde in 50 mL of ethanol. The mixture wasrefluxed for 18 hours. A bright yellow solution was formed. The solventwas removed by rotary evaporation, leaving an oil. The oil wascrystallized from hot ethanol, yieldingbis(salicylidine)-4-(p-nitrobenzyl)diethylenetriamine, mp 86°-88° C. andfurther characterized by: ¹ H NMR (CDCl₃) δ 2.8-3 (t, 4H), 3.6-3.9 (m,6H), 6.8-8 (m, 12H), 8.2 (s, 2H). ¹³ C NMR (CDCl₃) δ 8 55.27, 57.76,58.68, 116.87, 118.55, 123.42, 128.90, 131.17, 132.20, 147.15, 160.97,165.85.

EXAMPLE D

Preparation of 4-(p-aminobenzyl)diethylenetriamine

In 60 mL of absolute ethanol was dissolved 1 g of4-(p-nitrobenzyl)diethylenetriamine (prepared in Example B) and 100 mgof palladium on activated charcoal (10% Pd) was added. The mixture washydrogenated at 40 psi (275.8 kPa) for 24 hours. The catalyst wasremoved by filtration and the filtrate evaporated to give 0.9 g ofproduct as an oil. The oil solidified upon cooling and the solid wascrystallized from ethanol to provide4-(p-aminobenzyl)-diethylenetriamine and further characterized by: ¹ HNMR (CDCl₃) δ 8 2.2-2.8 (m, 12H), 3.4 (s, 2H), 6.4-7.15 (m, 4H). ¹³ CNMR (CDCl₃) δ 38.05, 55.54, 57.01, 113.34, 121.28, 128.42, 143.0.

EXAMPLE E

Preparation of1,7-bis(pyrrolecarboxylidine)-4-(p-nitrobenzyl)diethylenetriamine.

(p-Nitrobenzyl)diethylenetriamine, 2 g (8.5 mmol), was dissolved in 150mL of ethanol and 1.6 g (17 mmol) of (2-pyrrole)carboxaldehyde wasadded. The mixture was refluxed for 16 hours. The ethanol wasevaporated. The product,1,7-bis(pyrrolecarboxylidine)-4-(p-nitrobenzyl)-diethylenetriamine, wasobtained as an oil.

The product,1,7-bis(pyrrolecarboxylidine)-4-(p-nitrobenzyl)diethylenetriamine, wasused without purification or characterization in the following examples.

EXAMPLE F

Preparation of α,α'-dithiodiisobutyraldehyde.

A modified procedure by Niederhauser (U.S. Pat. No. 2,580,695) wasemployed. To a stirred solution of 578 g (8.0 mol) of isobutyraldehydein 920 g of carbon tetrachloride, was added dropwise at 40°-50° C. in 4hours 540 g (4.0 mol) of 98% sulfur monochloride. Hydrogen chloride gaswas copiously liberated and occasional cooling was necessary during theaddition period. The stirred solution was held at 30°-40° C. for anadditional 48 hours while a current of nitrogen was passed through thesolution in order to remove the hydrogen chloride. The solution wasdistilled and an 80% yield of α,α'-dithiodiisobutyraldehyde was obtainedand further characterized by: ¹ H NMR (CDCl₃) δ 1.40 (s, 12H), 9.09 (s,2H).

EXAMPLE G

Preparation of3,3,13,13-tetramethyl-1,2-dithia-5,8,11-triazatridecane-8-(p-nitrobenzyl)-5,11-diene.

To a stirred solution of 1.24 g (6.025 mmol)α,α'-dithiodiisobutyraldehyde (prepared in Example F) in 70 mL ofanhydrous benzene was added dropwise 1 g (6.025 mmol) of4-(p-nitrobenzyl)diethylenetriamine (prepared in Example B). The mixturewas refluxed for 16 hours and the reaction monitored by thin layerchromatographry (TLC) (ether/hexane, 1/1, v/v). After completion of thereaction, the solution was transferred to a separatory funnel and washedseveral times with water. The organic layer was dried over magnesiumsulfate and the solvent evaporated. The Shiff base was obtained as aglassy residue and was used without further purification andcharacterized by: ¹ H NMR (CDCl₃) δ 1.5 (s, 12H), 2.8 (t, 4H), 3.48 (t,4H), 3.78 (s, 2H), 7.6 (d, 2H), 8.2 (d, 2H); ¹³ C NMR (CDCl₃) δ 26.09,55.01, 58.78, 59.85, 123.52, 128.49, 129.09, 129.45, 168.15.

PREPARATION OF LIGAND FINAL PRODUCTS EXAMPLE 1

Preparation of bis(2-hydroxybenzyl)-4-(p-nitrobenzyl)diethylenetriamine.

In 70 mL of ethanol was dissolved 2.0 g (4.5 mmol) ofbis(salicylidine)-4-(p-nitrobenzyl)diethylenetriamine (prepared inExample C). To the solution was added 0.3 g (8.1 mmol) of sodiumborohydride. The reaction mixture was stirred for 2 hours at roomtemperature. A white solid precipitated, was filtered, and washed withaqueous ethanol. The product was recrystallized from absolute ethanol toprovide bis(2-hydroxybenzyl)-4-(p-nitrobenzyl)diethylenetriamine, mp108°-110° C. and further characterized by: ¹ H NMR (CDCl₃) δ 2.6 (s,8H), 3.6 (s, 2H), 3.9 (s, 4H), 6.3 (b, 2H), 6.7-8.2 (m, 14H). ¹³ C NMR(CDCl₃) δ 46.01, 52.45, 54.08, 58.85, 116.27, 118.98, 122.18, 123.69,128.24, 128.73, 129.33, 146.61, 158.04.

EXAMPLE 2

Preparation of bis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine.

In 40 mL of absolute ethanol was dissolved 400 mg ofbis(2-hydroxybenzyl)-4-(p-nitrobenzyl)diethylenetriamine (prepared inExample 1) and 100 mg of palladium on activated charcoal (10% Pd). Themixture was hydrogenated at 40 psi (275.8 kPa) for 24 hours. Thecatalyst was removed by filtration. The ethanolic solution wasevaporated to providebis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine, and wascharacterized by: ¹ H NMR (CDCl₃), carbon protons only; amine andhydroxyl protons were not included: δ 2.6 (s, 8H), 3.4 (s, 2H), 3.8 (s,4H), 6.4-7.2 (m, 12H). ¹³ C NMR (CDCl₃) δ 46.06, 52.35, 53.48, 58.74,115.08, 116.33, 118.87, 122.50, 128.35, 128.62, 130.03, 145.74, 158.31.

EXAMPLE 3

Preparation of3,3,11,11-tetramethyl-4,7,10-triazatridecane-7-(p-aminobenzyl)-2,12-dionedioxime.

To a dry flask was added 0.49 g (2.35 mmol) of4-(p-aminobenzyl)diethylenetriamine and 100 mL of dry methanol. Thesolution was cooled at 0° C. To the solution was added 0.64 g (4.7 mmol)of 3-chloro-3-methyl-2-butanone oxime. The solution was stirred at 0° C.for 1.5 hours and then refluxed for 16 hours. After the mixture wascooled, the volatiles were removed by rotary evaporation. A yellowglassy solid was neutralized with Na₂ CO₃ solution. The resulting gummysolid was extracted with two 100 mL portions of diethyl ether. The etherwas removed by rotary evaporation. The product was loaded onto a silicagel column and eluted with chloroform. The third band eluted was takenas the product and gave as an oil, after extraction with chloroform androtary evaporation,3,3,11,11-tetramethyl-4,7,10-triazatridecane-7-(p-aminobenzyl)-2,12-dionedioxime,which was characterized by: ¹ H NMR (CDCl₃) δ 1.7 (s, 12 H), 1.9 (s,6H), 3.1 (s, 6 H), 3.3 (s, 4H), 4.3 (s, 2H), 6.5-7.3 (m, 6H), 9.3 (b,2H).

EXAMPLE 4

Preparation of1,7-bis(2-methylenepyrrole)-4-(p-nitrobenzyl)diethylenetriamine.

In 150 mL of ethanol was dissolved 2 g of1,7-bis(pyrrolecarboxylidine)-4-(p-nitrobenzyl)diethylenetriamine(prepared in Example E) and 2 g of sodium borohydride was added. Thereaction mixture was stirred at room temperature for 2 hours. Thesolvent was evaporated and the residue was dissolved in 200 mL of water.The product was extracted in chloroform twice (150 mL portions). Anytrace of water was removed from the extract by the addition of about 40g of anhydrous sodium sulphate. The product was filtered and thechloroform removed by rotary evaporation to provide1,7-bis(2-methylenepyrrole)-4-(p-nitrobenzyl)diethylenetriamine and wascharacterized by: ¹ H NMR (CDCl₃), carbon protons only; amine andpyrrole protons were not included: δ 2.5 (s, 8H), 3.6 (s, 2H), 3.8 (s,4H), 6.1 (m, 6H), 7.4-8.1 (m, 4H).

EXAMPLE 5

Preparation of1,7-bis(2-methylenepyrrole)-4-(p-aminobenzyl)diethylenetriamine.

Reduction of 300 mg of1,7-bis(2-methylenepyrrole)-4-(p-nitrobenzyl)diethylenetriamine(prepared in Example 4) in 100 mL of ethanol was preformed using 100 mgof palladium on activated charcoal (10% Pd). The mixture washydrogenated at 48 psi (331 kPa) for 48 hours. The catalyst was removedby filtration. The ethanolic solution was evaporated to provide1,7-bis(2-methylenepyrrole)-4-(p-aminobenzyl)diethylenetriamine, and wascharacterized by: ¹ H NMR (CDCl₃), carbon protons only; amine andpyrrole protons were not included: Free amine: δ 2.5-2.7 (m, 8H), 3.5(s, 2H), 3.7 (s, 4H), 6-6.2 (m, 3H), 6.5-6.7 (m, 3H), 6.9-7.2 (m, 4H);HCl salt: δ 6.7-7.0 (2d, 4H), 3.35-3.55 (broad s, 12H), 3.25 (s, 2H),1.49 (s, 12H).

EXAMPLE 6

Preparation of2,2,12,12-tetramethyl-4,7,10-triaza-7-(p-aminobenzyl)-1,3-tridecanedithiol.

The crude2,2,12,12-tetramethyl-4,7,10-triaza7-(p-aminobenzyl)-1,13-tridecadiene-1,13-dithiol(prepared in Example G), 2.75 g (0.00671 mol) was dissolved in 25 mL ofdry tetrahydrofuran and added dropwise with cooling, under a nitrogenatmosphere, to a stirred suspension of (0.02 mol) of lithium aluminiumhydride. The mixture was heated under reflux for 40 hours andsubsequently hydrolyzed by the cautious addition of about 50-75 mL ofsaturated sodium and potassium tartarate solution. To the mixture wasthen added 300 mL of diethyl ether. The sludge was separated bydecanting or filtration (celite) and washed well with diethyl ether.Subsequently the solution was washed with brine and the organic layerwas separated and dried over sodium sulfate. The solvent was cautiouslyevaporated to dryness. The residue was dissolved in anhydrous diethylether and treated with hydrogen chloride gas. The sticky productprecipitated as the hydrochloride salt and was characterized by: ¹ H NMR(CDCl₃) δ 1.51 (s, 12H), 2.65 (t, 4H), 3.29-3.68 (m, 1OH), 6.6-6.9 (2d,4H). ¹³ C NMR (CDCl₃) δ 27.43, 48.23, 53.25, 54.13, 58.43, 59.17,122.67, 128.05, 129.71, 144.5.

PREPARATION OF COMPLEX AND CONJUGATE FINAL PRODUCTS EXAMPLE 7

Complexation of ¹⁰⁵ Rh withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine and theconjugation to IgG.

A. Complexation of ¹⁰⁵ Rh withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine

Complexation of bis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine(prepared in Example 2) with rhodium was tried at differentconcentrations of the ligand as well as carrier rhodium. Typically, 0.5mL of sodium bicarbonate buffer (0.5M, pH 9, 0.1 mL) and ¹⁰⁵ Rh (15 MBq)and 0.05 mL of RhCl₃ (1.25×10⁻³ mmol) (i.e. carrier rhodium) were mixedtogether and refluxed for about 10 min in a 10 mL round bottom flaskfitted with a glass condenser over a boiling water bath. To thisreaction mixture was added 0.5 mL (1.5×10⁻³ mmol) of an ethanolicsolution of the ligand. The mixture was then further refluxed for 2hours and the mixture turned yellow.

The solution was cooled to room temperature and transferred to a 10 mLcentrifuge tube. Upon cooling, the solution turns green. After a 10 μLaliquot (first) was removed, the remaining solution was centrifuged for15 min. The clear supernatant solution was transferred to another vial.A 10 μL aliquot (second) was removed and counted for radioactivity (in aNaI(T1) scintillation counter after suitable modification of thegeometry). By comparing the activity of the second aliquot with theactivity in the first aliquot, the amount of activity lost as sedimentswas calculated. The final concentration of Rh in solution was calculatedby taking into account this loss of activity and the total volume afterheating.

Complex yields were estimated by an MgO adsorption technique. Thismethod is based on the observation that inorganic rhodium complexes areadsorbed by MgO leaving behind the organic complexes in solution.Typically, 10 μL of the complex solution was diluted to 0.4 mL and about50 mg of MgO powder was added and mixed over a vortex mixer for 2 minfollowed by a 5 min centrifugation. The supernatant was separated andboth fractions counted. The activity associated with the supernatant wasan estimate of the complex present.

The results of the complexation are shown in Table 1 which shows thecomplex prepared with different Rh to ligand ratios. The ligand/metalratio varied from 1 to 5 in the experiments with carrier rhodium. In theexperiment where there was no carrier added rhodium, the ligand ispresent in a concentration at about 10⁶ higher than rhodium.

The amount of rhodium activity lost as sediments varied from 5-15%.Column 6 in Table 1 shows the yield estimated by MgO adsorptiontechnique. The complex yield in solution after removal of sedimentsvaried between 85-91%. The overall complex yields were calculated bytaking into account the activity in solution and complex yield insolution as estimated by MgO adsorption technique. The overall complexyields, calculated by multiplying the activity yield in solution bycomplex yields, were between 75-83%.

                                      TABLE 1                                     __________________________________________________________________________                    %.sup.b                                                                            %.sup.c   %.sup.e                                                        Activity                                                                           Complex                                                                            %.sup.d                                                                            Activity                                                                            %.sup.f                                                                            %.sup.g                                 [Rh]                                                                              [L]     in   in   Complex                                                                            in    Conjuga-                                                                           Overall                             RUN × 10.sup.4a                                                                 × 10.sup.3a                                                                 L/Rh                                                                              solution                                                                           solution                                                                           yield                                                                              chloroform                                                                          tion yield                                                                              Rh/IgG.sup.h                   __________________________________________________________________________    1   12.5                                                                              1.25                                                                              1   85   91   78   82    87   61   1.7                            2   5   1.25                                                                              2.5 91   90   82   85    92   71   0.8                            3   2.5 1.25                                                                              5   88   85   75   79    90   62   0.4                            4   NCA 12.5                                                                              NCA 95   87   83   76    92   66   NCA                            __________________________________________________________________________     L is bis(2hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine                  NCA means No Carrier Added                                                    .sup.a total volume of reaction 2.1 mL (e.g. Run 1: Rh = 12.5 ×         10.sup.-4 M, L = 1.25 × 10.sup.-3 M)                                    .sup.b % of total activity in solution                                        .sup.c % of which complexed                                                   .sup.d % of total activity converted to complex (e.g. Run 1: 0.85 ×     .91 = 0.78)                                                                   .sup.e % of activity in solution extracted into chloroform after              conversion to isothiocyanate                                                  .sup.f % of activity extracted into chloroform conjugated to protein          .sup.g overall yield = % activity in solution (b) × % activity in       chloroform (e) × % activity in conjugation (f)                          .sup.h the average number of Rh atoms per IgG molecule                   

In table 1, conjugation reactions were run such that [L] (based on data)(a)/[IgG] was always 2.8 and the atoms of Rh/molecule IgG=2.8×(g)×(Rh/Lfrom(a)). For example, Run 1: 2.8×0. 61×1=1.7; and Run 2:2.8×0.71×1/2.5=0.8.

Table 2 gives the complexation yield studied for the reaction carriedout at different concentration ofbis-(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine and rhodium.The rhodium to bis-(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamineratio was kept constant at 1 except for the reaction with no carrieradded rhodium. The complexation reaction was clean in all these caseswith better than 96% of the activity in solution after centrifugation.The complex yield in solution varied between 87-94%. Overall complexyields varied between 85-90%. In Table 2, the conjugation reactions wererun such that the ligand (L), based on data) (a)/IgG was always 1.12.Calculations were the same as for Table 1.

                                      TABLE 2                                     __________________________________________________________________________                    %.sup.b                                                                            %.sup.c   %.sup.e                                                        Activity                                                                           Complex                                                                            %.sup.d                                                                            Activity                                                                            %.sup.f                                                                            %.sup.g                                 [Rh]                                                                              [L]     in   in   Complex                                                                            in    Conjuga-                                                                           Overall                             RUN × 10.sup.4a                                                                 × 10.sup.4a                                                                 L/Rh                                                                              solution                                                                           solution                                                                           yield                                                                              chloroform                                                                          tion yield                                                                              Rh/IgG.sup.h                   __________________________________________________________________________    1   12.5                                                                              12.5                                                                              1    96  94   90   84    86   70   0.8                            2   5   5   1    96  89   85   83    93   75   0.8                            3   2.5 2.5 1   100  87   87   82    90   74   0.8                            4   NCA 2.5 NCA 100  88   87   69    90   62   NCA                            __________________________________________________________________________     L is bis(2hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine                  NCA means No Carrier Added                                                    .sup.a total volume of reaction 2.1 mL (e.g. Run 1: Rh = 12.5 ×         10.sup.-4 M, L = 1.25 × .sup.-3 M)                                      .sup.b % of total activity in solution                                        .sup.c % of which complexed                                                   .sup.d % of total activity converted to complex (e.g. Run 1: 0.96             .sup.e % of activity in solution extracted into chloroform after              conversion to isothiocyanate                                                  .sup.f % of activity extracted into chloroform conjugated to protein          .sup.g overall yield = % activity in solution (b) × % activity in       chloroform (e) × % activity in conjugation (f)                          .sup.h the average number of Rh atoms per IgG molecule                   

In Table 2, the conjugation reactions were in a manner analogous to thatof Table 1.

The extractability of the complex in chloroform was estimated. Thisvaried from 20-35% in different batches. A back extraction of thecomplex from the organic layer into saline gave 67% of the activitystill remaining in the organic layer. However, the second extraction ofthe aqueous layer gave only 9% of the activity in the organic layer.These results suggests the possibility of the presence of more than onespecies of complex in solution.

Tables 1 and 2 give the activity transfer into the organic layer duringthe reaction with thiophosgene. Note that only 0.1 mL of chloroform wasused for extraction as against 1 mL of complex solution. The majority ofthe activity was transferred into the organic layer. As theextractability of the complex itself was not very high, the higherextractability seen after activation was assumed to be due to the higherpartition coefficient of the activated complex. The partitioncoefficient of the isothiocyanate derivative of the complex wasestimated by repeated back extractions of it into saline and was foundto be around 60.

B. Preparation of the isothiocyanate derivative.

The isothiocyanate derivative of the complex from Part A above wasprepared by treating an aqueous solution of the complex with an excessconcentration of thiophosgene in chloroform. The isothiocyanatederivative of the complex is referred to as the activated complex.

Typically 1 mL of the complex (about 7×10⁻⁴ mmol) prepared above wasmixed for 2 min with 0.1 mL (1.3×10-2 mmol) of thiophosgene, diluted inchloroform, over a vortex mixer. The majority of the activity, which wasbelieved to be due to the activated complex, was transferred into theorganic layer. The aqueous layer was carefully withdrawn into anothertube. The amount of activity transferred into the organic layer iscalculated by counting a 10 μL aliquot of the aqueous layer before andafter activation. The organic layer was dried under a stream of nitrogengas to remove any chloroform and unreacted thiophosgene. The driedactivated complex was dissolved into 100 μL dimethylformamide and usedfor conjugation.

C. Conjugation.

IgG, 2 mL, dissolved in 0.1M borate buffer, pH 9 containing 0.15M NaCl,was mixed with 20 μL (10⁻⁴ mmol) of the activated complex indimethylformamide. The conjugation reaction was carried out at roomtemperature for 4-5 hours.

D. Estimation of conjugation yields.

The conjugation yields were estimated by gel permeation chromatography.A 30×1.4 cm column was packed with presoaked Sephedex™ G 75 gel andequilibrated by passing 100 mL of 0.15M NaCl solution through thecolumn. The reaction mixture, 0.1 mL, was applied to the top of thecolumn and eluted with 0.15M NaCl solution. Fractions of 2 mL each werecollected and the activity measured in a NaI(T1) scintillation counter.Recovery from the column was monitored by counting an equal aliquot,after dilution to 2 mL, and comparing it with the sum of the activityeluted from the column. Activity associated with the protein peak wassummed and compared to the total activity for the estimate of the yieldof conjugation.

Conjugation studies, with complex prepared at differentbis-(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine to Rh ratiosand also at different concentrations of Rh, gave high conjugation yields(Tables 1 and 2 before). Yields varied from 86-93% and was found to beindependent of the complexation condition. In conjugation experimentswith differentbis-(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine/Rh ratios, thereaction was performed with the ligand:IgG ratio at 2.8:1. Note that theligand concentrations used are based on the data (a). The actual amountof ligand still present at the time of conjugation is not known. Theequations given for Table 1 convert the ratio to the Rh/IgG actuallyconjugated. In these studies since excess ligand was used forcomplexation all the ligand was not utilized for complexation and hencethere is free ligand present. The number of rhodium atoms incorporatedin these studies varied from 0.4-1.7 depending upon the starting Rhratio. The overall rhodium recovery at the end of the conjugationreaction varied from 61 to 71% without taking into account the activityloss due to decay.

In the conjugation experiments withRh/bis-(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine ratio keptconstant at 1 (Table 2), the number of Rh atoms incorporated was from0.8-1.12. Note that the ligand/IgG ratio was kept constant at 1.12 inthese conjugation experiments. The final rhodium recovery in theseexperiments varied from 70-75%.

Table 3 gives the results of conjugation studies with different Rh toIgG ratios while keeping the IgG concentration constant and varying thecomplex concentration. The complex used in this study was prepared at1:1Rh/Ligand and the complex yield was 95%. The conjugation yield variedfrom 85-93% and we could incorporate 0.5-8.5 mmols of Rh per mmol ofantibody. The overall Rh yield in these studies varied from 69-75%taking into account activity lost in all steps.

                  TABLE 3                                                         ______________________________________                                                                Conjuga-       %                                      [IgG] [Rh]              tion    Rh/IgG.sup.a                                                                         Rhodium.sup.b                          × 10.sup.5                                                                    × 10.sup.5                                                                       Rh/IgG   %       Labeled                                                                              Yield                                  ______________________________________                                        5     2.5      0.5      91      0.5    74                                     5     5        1        93      0.9    75                                     5     10       2        93      1.9    75                                     5     25       5        92      4.6    74                                     5     50       10       85      8.5    69                                     ______________________________________                                         .sup.a This is the number of rhodium atoms incorporated per molecule of       IgG                                                                           .sup.b Calculated by taking into account activity lost as sediments, lost     aqueous layer and the conjugation yield.                                 

Table 4 gives the results of conjugation yield studied with differentratios of protein and complex. The concentration of complex is keptconstant and the concentration of IgG varied with Rh/IgG from 1-10. Itwas seen that the yield decreased from 91-73% when IgG concentration wasreduced from 5×10⁻⁵ to 5×10⁻⁶ mmol. The number of Rh/atoms incorporatedper IgG molecule varied from 0.9-7.3 in these experiments. These resultssuggest that there is greater dependence on the concentration of IgGthan that of the activated complex for conjugation.

                  TABLE 4                                                         ______________________________________                                                                Conjuga-       %                                      [IgG] [Rh]              tion    Rh/IgG.sup.a                                                                         Rhodium.sup.b                          × 10.sup.6                                                                    × 10.sup.5                                                                       Rh/IgG   %       Labeled                                                                              Yield                                  ______________________________________                                        50    5        1        91      0.9    74                                     20    5        2.5      87      1.8    70                                     10    5        5        84      14.2   68                                     50    5        10       73      7.3    69                                     ______________________________________                                         .sup.a This is the number of rhodium atoms incorporated per molecule of       IgG                                                                           .sup.b Calculated by taking into account activity lost as sediments, lost     aqueous layer and the conjugation yield.                                 

E. EDTA challenge.

The purified protein, 2 mL, equivalent to 4.5×10⁻⁵ mmol of the complex,was mixed with 20 μL of 0.1M EDTA solution and then incubated for 24hours. The activity associated with the protein and EDTA fractions wasestimated by gel permeation chromatography.

The results of the EDTA challenge studies show that Rh complex is highlyinert and cannot be exchanged with other ligands at room temperatureeven at very high concentration of competing ligands. After a 24 hourschallenge study, 92% of the activity was still seen with the proteinpeak on chromatography. No separate peak was seen for EDTA or freecomplex when labeled protein solution challenged with EDTA wasrechromatographed.

F. Affinity chromatography.

Affinity chromatography was done on an anti-IgG agarose gel column. TwomL of the gel was packed into a syringe column and equilibrated with 20mL of 0.01M phosphate buffer, pH 7.2. The solution from the conjugatereaction mixture, 50 μL, was applied to the top of the column.Non-immunoreactive fractions were collected in 10 mL of phosphatebuffer. Antibody bound IgG was eluted with 0.05M acetic acid containing0.2M sodium acetate, pH 2.5. Fractions, 1 mL, were collected andactivity monitored on a NaI (T1) gamma scintillation counter.

Affinity chromatography of the labeled IgG from Run 3, Table 2 gave 81%of the activity retention in the affinity column as against 90% yield ofconjugation. This indicates that 90% of the labeled IgG retainedimmunoreactivity after conjugation. Labeled proteins from all the runswere not studied by affinity chromatography, instead an affinityadsorption study was done which is less time consuming and has theadvantage that multiple samples can be handled. However, the actualpercentage of immunoreactive component present is not given but only anindex. The results of affinity adsorption studies are shown in Table 5.Labeled IgG prepared in different batches showed greater than 61%adsorption to the affinity gel. The unactivated complex ofbis-(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine was run withthe affinity gel under similar conditions and found that less than 5% ofthe activity was retained in the affinity gel after the third wash.

                  TABLE 5                                                         ______________________________________                                        Table       Run    Adsorption on affinity gel                                 ______________________________________                                        1           1      60                                                         1           2      63                                                         1           3      66                                                         1           4      60                                                         2           1      60                                                         2           2      74                                                         2           3      77                                                         2           4      71                                                         ______________________________________                                    

G. Affinity adsorbtion studies.

Affinity gel, 1 mL, was incubated with 25 μL (about 0.2 mg of IgG) ofthe solution from the conjugation reaction for 15 min with gentleshaking. The gel was washed (3 times, 2 mL) with phosphate buffer toremove unbound activity. After the third wash the gel was counted forradioactivity. The original solution, 25 μL was also counted forradioactivity under identical conditions. The percentage retention ofactivity to the gel was calculated by comparing the activity in the gelwith total activity.

H. Conjugation kinetics.

Time dependence of the conjugation reaction was studied by incubating 80μL of the activated complex (1.5×10⁻⁴ mmol) with 3 mL of IgG solution(5×10⁻⁵ M). This solution, 100 μL , was withdrawn at various timeintervals and conjugation yield estimated by gel permeationchromatography.

I. Stability of activated complex.

The chloroform extract of the activated complex, 40 μL, (about 10⁻⁴mmol) was dispensed into tubes and dried under nitrogen. The tubes weresealed and stored at room temperature. One tube was taken at differenttime intervals and the contents dissolved in 50 μL of dimethylformamide.Then a 25 μL aliquot of the solution was added to 1 mL of IgG solution(5×10⁻⁵ mmol). The conjugation reaction was carried out for 5 hours andthe yield estimated by gel permeation chromatography.

Table 6 gives the results of conjugation studies carried out withactivated complex stored for different time intervals. No significantdifference was seen with activated complex stored up to 4 days onconjugation.

                  TABLE 6                                                         ______________________________________                                        Time (hours)  Conjugation %                                                   ______________________________________                                         0            90                                                              24            89                                                              48            90                                                              72            94                                                              96            92                                                              ______________________________________                                    

EXAMPLE 8

Complexation of ^(99m) Tc withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine and theconjugation to IgG.

A. Complexation of ^(99m) Tc withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine.

The complex of ^(99m) Tc withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine (prepared inExample 2) was prepared using saturated tin tartarate solution as thereducing agent. Before adding the tin tartarate solution to thecomplexation mixture, it was purged with nitrogen and centrifuged toremove solid particles. The complexation mixture was then prepared from0.5 mL of bicarbonate buffer (0.5M, pH 9), 0.2 mL ofbis(2-hydroxybenzyl)-4-(p-aminobenzyl)-diethylenetriamine (5×10⁻⁴ mmol)in ethanol, 1 mL ^(99m) Tc (37-74 MBq) and 2.6 mL of 0.9% salinesolution. The complexation mixture was mixed over a magnetic stirrer. Tothis mixture was added 0.2 mL of the tin tartarate solution and themixture was mixed over a magnetic stirrer for 10 min.

B. Paper Chromatography and thin layer chromatography.

To two 1×13.7 cm solvent saturation pads (Gelman Sciences Inc.) wasapplied 5 μL of the complex solution (prepared in Example 7A). The padswere developed in acetone and 0.9% saline. After drying the solvents,the papers were cut into 6 sections and counted for radioactivity. Thewhole chromatograph was divided into 2 parts--from the point of spottingto the middle of the paper and from the middle of the paper to thesolvent front--and the percentage of activity associated with these twofractions computed.

Thin layer chromatography was carried out on 2.5×7.5 cm flexible silicagel plates. The solution (5 μL) was spotted about 1 cm from the end ofthe plate and then developed in saline up to 7 cm. The plates wereremoved, dried and cut into 10 strips of equal size. The strips werecounted for radioactivity in a well-type NaI (T1) counter. Percentagesof the complex activity at the point of spotting and solvent front werecalculated.

The distributions of complex activity in paper and thin layerchromatography is given in Table 7. In using paper chromatography, withboth acetone and saline as eluents, 75-80% of the activity was seen atthe solvent front. In thin layer chromatography using saline as eluent,19% of the activity was seen at the solvent front. In controlexperiments, TcO₄ ⁻ was seen at solvent front in thin layerchromatography/saline.

                  TABLE 7                                                         ______________________________________                                                      Distribution of Activity                                        Method          Rf = 0    Rf = 1                                              ______________________________________                                        PC/SALINE       21        79                                                  PC/ACETONE      24        76                                                  TLC/SALINE      81        19                                                  ______________________________________                                    

In Table 2, PC means paper chromatography, and TLC means thin layerchromatography.

C. HPLC (High Performance Liquid Chromatography)

HPLC analyses were done in Beckman model 332 gradient liquidchromatographic system. Eluent radioactivity was measured by Beckman™model 170 radioisotope detector. A Hamilton™ PRP-1 reverse phase column(30.5 cm) was used for complex analysis. Samples of 15 μL were injectedinto the column. A gradient elution using water (A) and acetonitrile (B)was used with a flow rate of 2 mL/min. Typically the flow pattern was:

concentration of B in A

0-2 min 10% B

2-4 min linear gradient of 70% B

4-18 min 70% B

38-40 min 10% B linear gradient

After completion of each run, the column was equilibrated for 10 minwith 90% A and 10% B solution. Between each run the injection port wasflushed with methanol. When the column was not in use it was kept in 9/1B/A. TcO₄ ⁻ was eluted at R_(t) =1.0 min. and the complex at R_(t)=7.0-7.5 min. Complex was eluted mainly as a single peak. TcO₄ ⁻estimated by this method was about 20%.

D. Solvent Extraction

The lipophilic complex formed was extracted into chloroform. Thereaction mixture, 4 mL, was mixed with 4 mL of chloroform and furthermixed with a vortex mixer for 2 min. The organic and aqueous layers wereseparated. A second extraction was carried out by mixing 1 mL of theaqueous layer with 1 mL of chloroform and extracted as before. One mL ofthe organic layer from the first extraction was back extracted with 1 mLof 0.1M NaHCO₃ buffer, pH 9.0. The partition coefficient was calculatedfrom the back extraction data.

The complex showed very high extractability in chloroform. Theextraction yield in different batches varied from 85-95%. The secondextraction yield was generally low, 10% of the remaining activity. Thechloroform/buffer partition coefficient was about 90. This highpartition coefficient enables quantitative separation of the complex bya single extraction.

The complexation reaction is complete in 5-10 min. About 95% of theactivity could be extracted into chloroform for the first one hour ofcomplexation. Prolonged reaction resulted in less extractable activity.After a 24 hour reaction the solvent extractable activity was only 55%.

E. Preparation of the isothiocyanate derivative.

To 3 mL of the organic layer (3×10⁻⁴ mmol of the ligand) was added 0.5mL of thiophosgene (3.3×10⁻²) diluted in chloroform and the the mixturestirred for 2 min. Excess thiophosgene and chloroform were removed byevaporation under a flow of nitrogen. The activated complex was thendissolved into 0.6 mL of dimethylformamide to give a ligandconcentration of 5×10⁻⁴ mmol/mL.

F. Conjugation to protein.

Conjugation of the activated complex from part E above to protein (IgG)was carried out in 0.1M borate buffer, pH 9.0 containing 0.9% saline. To1 mL IgG (5×10⁻⁵ mmol) was added activated complex solution, varyingfrom 5×10⁻⁶ to 10⁻⁴ mmol, and the solution was incubated for 2-4 hours.The ligand/IgG ratio varied from 0.1 to 2.0. Note that those ratios arebased on the amount of ligand used to form the complex. Since theconcentration of ^(99m) Tc is so much lower than the ligand, the ligandconcentrations are shown only to demonstrate the affects of amounts ofcomplex on the conjugation.

In an alternate experiment, ligand concentration was maintained constantat 5×10⁻⁵ mmol/mL. IgG concentration was varied from 5×10⁻⁵ to 10⁻⁴mmol/mL. The reaction volume in this instance was 0.25 mL. A blankexperiment was carried out by incubating 0.5 mL of the unactivatedcomplex (5×10⁻⁵ mmol) with 1 mL of IgG solution (5×10⁻⁵ mmol) for 4hours.

G. Estimation of conjugation yields.

Conjugation yields were estimated by gel permeation chromatography overSephadex™ G 75 column (30×1.4 cm). An aliquot of the reaction mixturewas applied on the top of the column and eluted with 0.9% salinesolution. Fractions, 2 mL, were collected and the activity measured in aNaI (T1) scintillation counter. An equal amount of the sample applied tothe column was diluted to 2 mL and kept as a control. Recovery from thecolumn was calculated by comparing the sum of activity eluted from thecolumn to the activity in the control tube. The conjugation yield wascalculated by taking the ratio of the sum of the protein peak to thetotal recovered activity.

Table 8 shows the conjugation yields when IgG concentration is keptconstant and the ligand concentration varied to give differentcomplex/IgG ratios. The conjugation yield was 67% at a ratio of 0.1complex/IgG but remained constant around 79% from 0.5 to 2.0. The lowyield seen at the ratio of 0.1 complex/IgG may be due to the slowerkinetics of these reactions at this concentration.

                  TABLE 8                                                         ______________________________________                                        [IgG]   [Complex]                                                             × 10.sup.5                                                                      × 10.sup.6                                                                           Complex/IgG % Yield                                      ______________________________________                                        5        5           0.1         67                                           5       25           0.5         79                                           5       50           1.0         80                                           5       100          2.0         79                                           ______________________________________                                    

Table 9 gives conjugation yield when complex concentration is keptconstant and IgG concentration varied from 1 to 5. At the lowestconcentration of protein (10⁻⁵), the yield was >70%. The amount ofprotein used at this level was 375 μg.

                  TABLE 9                                                         ______________________________________                                        [IgG]   [Complex]                                                             × 10.sup.5                                                                      × 10.sup.5                                                                           Complex/IgG % Yield                                      ______________________________________                                        5       5            1           73                                           2.5     5            2           74                                           1.0     5            5           71                                           ______________________________________                                    

When unactivated complex was incubated with IgG and chromatographed in aSephadex™ column, only 3% of the activity was seen with the protein. Therest of the activity was eluted between 30 and 50 mL.

H. EDTA challenge.

A 2 mL fraction of the protein peak, isolated from the gel permeationchromatography, was incubated with 10 μL of 10⁻¹ M EDTA solution andincubated for 24 hours. The EDTA/ligand ratio was 200 and 400 in the twosets of experiments. Activity associated with EDTA and protein wasestimated by gel permeation chromatography as described before.

Results of the EDTA challenge studies are given in Table 10. Recoveryfrom the gel permeation column was 84%. More than 95% of the recoveredactivity was seen with IgG.

                  TABLE 10                                                        ______________________________________                                                 % Recovery                                                           EDTA/    from the      % Activity                                                                              % Activity                                   Complex  column        with IgG  with EDTA                                    ______________________________________                                        200      84            82        2                                            400      84            80        4                                            ______________________________________                                    

EXAMPLE 9 Complexation of ⁵⁷ Co withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine and theconjugation to IgG.

A. Complexation of ⁵⁷ Co withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine.

A 10⁻² mmol solution of Co(II) was prepared by dissolving Co(II)perchlorate in saline. 0.1 mL of this solution was spiked 2 μL of ⁵⁷ Co(about 20 μCi) and equilibrated for 24 hours.

Complexation was carried out by mixing 100 μL (2.5×10⁻⁴) mmol) ofbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine (prepared inExample 2), with 10 μL of Co(II) solution (10⁻⁴ mmol) and incubating forhour. Complex yield was estimated by thin layer chromatography usingsaline as eluent. Complex has an R_(f) =0 free Co(II) has an R_(f) =1.0.The results of the complexation reaction are shown in Table 11.

                  TABLE 11                                                        ______________________________________                                        Ligand    Co(II)                                                              (mmol)    (mmol)     Ligand/Co(II)                                                                             % Complex                                    ______________________________________                                        10.sup.-4 10.sup.-4  1           18                                           2.5 × 10.sup.-4                                                                   10.sup.-4  2.5         97                                             5 × 10.sup.-4                                                                   10.sup.-4  5           97                                           ______________________________________                                    

Complexation yield was highly dependent on the ligand to Co(II) ratio.At ligand to Co(II) ratio greater than 2.5, complexation yield wasgreater than 97%.

B. Activation.

To the complex was added 5 μL (6×10⁻² mmol) of CSCl₂ and then thesolution was mixed for 2 min over a vortex mixer. The activated complexwas dried and the solid dissolved in 100 μL of dimethylformamide.

C. Conjugation.

The activated complex (2.5×10⁻⁵ -1.25×10⁻⁴ mmol) in dimethylformamidewas mixed with IgG (5×10⁻⁵ mmol) in borate solution and incubated for 24hours at room temperature. Conjugation yields were estimated by gelpermeation chromatography using a Sephedex™ G 75 column (30×1.4 cm).Saline solution (0.9%) was used as eluent. The results of theconjugation reaction are given in Table 12.

                  TABLE 12                                                        ______________________________________                                                 Activated                                                            IgG      complex                                                              (mmol)   (mmol)       Complex/IgG % Yield                                     ______________________________________                                        5 × 10.sup.-5                                                                    2.5 × 10.sup.-5                                                                      0.5         52                                          5 × 10.sup.-5                                                                      5 × 10.sup.-5                                                                      1           62                                          5 × 10.sup.-5                                                                    1.3 × 10.sup.-4                                                                      2.5         88                                          5 × 10.sup.-5                                                                    5 × 10.sup.-5a                                                                       1           40                                          5 × 10.sup.-5                                                                    5 × 10.sup.-5b                                                                       1           19                                          ______________________________________                                         .sup.a Unactivated complex used to estimate non specific labeling             .sup.b Co(II) solution used for estimating non specific labeling due to       free cobalt.                                                             

D. Non-specific labeling.

Non-specific labeling was estimated by using unactivated complex andCo(II) solution spiked with ⁵⁷ Co. In the former case, the complex wasdried and dissolved in 100 μL of dimethylformamide and 20 μL (5×10⁻⁵mmol) of this solution was incubated with 1 mL of IgG (5×10⁻⁵ mmol) for24 hours. In the latter case, 5 μL (5×10⁻⁵ mmol) of Co(II) solutionspiked with ⁵⁷ Co was incubated for 24 hours with 1 mL of IgG (5×10⁻⁵mmol). Non-specific labeling yields in both cases were estimated by gelpermeation chromatography. The non-specific labeling yields were highfor unactivated complex. See Table 12 above.

EXAMPLE 10

Complexation of ¹⁰⁵ Rh with1,7-bis(2-methylenepyrrole)-4-(p-aminobenzyl)diethylenetriamine and theconjugation to IgG.

A. Complexation of ¹⁰⁵ Rh with1,7-bis(2-methylenepyrrole)-4-(p-aminobenzyl)diethylenetriamine.

One mL of 1×10⁻³ M solution of RhCl₃ was spiked with 50 μL of ¹⁰⁵ Rh(about 120 μCi) and then warmed to attain equilibrium between thecarrier and active rhodium. To the solution was added 1.25×10⁻³ mmol (1mL) of 1,7-bis(2-methylene-pyrrole)-4-(p-aminobenzyl)diethylenetriamine(prepared in Example 5). This mixture was refluxed for 2 hrs.Complexation yield was estimated by the MgO method (as described inExample 7).

The complexation yield was independent of the ¹⁰⁵ Rh/ligand ratio as itwas always in the range of 77-80% even when carrier free ¹⁰⁵ Rh wasused.

B. Kinetics of the Complexation Reaction.

To determine the refluxing time needed for complete complexation, theyield was estimated at different refluxing times, 1-180 min, by the MgOmethod (as described in Example 6). The results of the kineticsexperiments are given in Table 13. The complex yield was found to reachits maximum after only 5 minutes of refluxing.

                  TABLE 13                                                        ______________________________________                                               Time (Min)                                                                            Yield %                                                        ______________________________________                                                1      72                                                                     5      79                                                                    10      79                                                                    30      79                                                                    60      78                                                                    180     75                                                             ______________________________________                                    

The percent of the total activity extracted into the organic phase wasabout 62%. The yield values in Table 11 above are based on the organicphase only.

C. Activation.

To 1 ml of the complex (prepared in Part B above) was added 1.3×10⁻²mmol CSCl₂ in 1 mL of chloroform. The solution was mixed for 2-3 min.The organic layer was separated and dried and the residue dissolved in150 μL of DMF.

D. Conjugation to protein.

The activated complex, 50 μl, (prepared in Part C) was added to 2 mL ofIgG (1×10⁻⁴ mmol) in borate buffer and then incubated for 24 hours atroom temperature. The conjugation yield was estimated by gel permeationchromatography.

Conjugation yield with IgG ranged from 88-97%. The ratio of complex toprotein was not determined because the coupling work was performed withcarrier free ¹⁰⁵ Rh. Overall yields then are (about 0.62×0.92) or about57%.

E. Blank

Unactivated complex was incubated with IgG for 24 hours at roomtemperature. The yield was estimated by gel permeation chromatography.The non-specific binding of the unactivated complex was 25-27%.

EXAMPLE 11

Complexation of ^(99m) Tc with2,2,12,12-tetramethyl-4,7,10-triaza-7-(p-aminobenzyl)-1,13-tridecanedithiol.

A. Complexation of ^(99m) Tc with2,2,12,12-tetramethyl-4,7,10-triaza-7-(p-aminobenzyl)-1,13-tridecanedithiol.

The complex of ^(99m) Tc with2,2,12,12-tetramethyl-4,7,10-triaza-7-(p-aminobenzyl)-1,13-tridecanedithiol (prepared in Example 6) was prepared by the stannous tartratereduction method. The complexation mixture was prepared from 0.5 mL ofbicarbonate buffer (0.5 mol, pH 9), 0.1 mL of2,2,12,12-tetramethyl-4,7,10-triaza-7-(p-aminobenzyl)-1,13-tridecanedithiol (5×10⁻³ M) in saline, 1 mL of ^(99m) Tc (as sodiumpertechnetate), 2.2 mL of 0.9% saline solution, and 1 mL of NH₄ TcO₄(1×10⁻⁷ M). To this mixture was added 0.2 mL of the saturated tintartrate solution and the mixture was allowed to stand for 15 min.

B. Paper Chromatography and Thin Layer Chromatrography

On two 1×13.7 cm solvent saturation pads (Gelman Sciences, Inc.) werespotted 10 μL of the complex from Part A above. The pads were developedin acetone and 0.9% saline. After drying the solvents, the papers werecut into 7 sections and counted for radioactivity. The wholechromatograph was divided into 2 parts, namely, from the point ofspotting to the middle of the paper and from the middle of the paper tothe solvent front. The percent of activity associated with these 2fronts were computed.

Thin layer chromatrography was carried out on 2.5×7.5 cm flexible silicagel plates. The complex solution, 5 μL, was spotted about 1 cm from theend of the plate and then developed in saline up to 7 cm. The plateswere removed, dried and cut out into 10 strips of equal size. The stripswere counted for radioactivity in a well type NaI (T1) counter.Percentages of the complex activity at the point of activity and thesolvent front were calculated.

In using paper chromatrography with acetone as the eluent, 80% of theactivity was seen at the solvent front. In thin layer chromatographyusing saline as the eluent, no activity was seen at the solvent frontindicating nearly 100% complexation, because in control experiments,TcO₄ ⁻ was seen at the solvent front in thin layer chromatography withsaline as the eluent.

C. High Performance Liquid Chromatograpgy (HPLC)

a. Equipment

HPLC analysis was done using a Beckman model 332 liquid chromatographicsystem. Eluent radioactivity was measured by a Beckman model 170radioisotope detector. A Hamilton PRP-1 reverse phase column was usedfor complex analysis.

b. Process

Samples from Part B above, 20 μL, were injected into the column. Asolvent system of methanol and water (80/20 v/v) was used as the elutingsolvents with a flow rate of 2 mL/min. TcO₄ ⁻ was eluted at R_(t) =1.98min. The complex was eluted at R_(t) =5.97 min. and was a single peak,indicating the presence of a single species. TcO₄ ⁻ estimated by thismethod was about 9%.

D. Solvent Extraction

The reaction mixture from Part C above, 1 mL, was mixed with 1 mL ofchloroform and vortexed for 2 min. The organic and aqueous layers wereseparated and counted. A second extraction was carried out by mixing 1mL of the aqueous layer with 1 mL of chloroform and extracted as before.The organic layer from the first extraction, 1 mL, was back extractedwith 1 mL of NaHCO₃ buffer, pH 9.0. The partition coefficient wascalculated from the back extraction data.

The complex was highly extractable in chloroform, with a yield in therange of from 80-90%, in studies performed in different batches. Thesecond extraction typically recovered only 8-10% of the remainingactivity. The partition coefficient in chloroform/buffer was about 90.This result facilitated a qunatative extraction of the complex by asingle extraction.

The complexation reaction was complete in 5-10 min. About 85% of theactivity could be extracted into chloroform in that time period. After24 hours the solvent extractable activity was about 78% for a singleextraction.

E. Preparation of the isothiocyanate derivative

To 0.5 mL (5×10⁻⁵ mmol) of the organic layer from Part D above was added20 μL (1.31×10⁻⁴ mmol) of thiophosgene and the reaction mixture stirredfor two min. Excess thiophosgene and chloroform were exaporated under astream of nitrogen. The activated complex was then dissolved in 100 μLof dimethylformamide to give 5×10⁻⁵ mmol of the activated complex.

F. Conjugation to Protein

Conjugation of the activated complex from Part E above to protein (IgG)was carried out in 0.1M borate buffer, pH 9.0 containing 0.9% saline.The activated complex was added to 1 mL of IgG (5×10⁻⁵ mmol) and thesolution incubated for 1 hour. Conjugation was thereby done at a complexto protein ratio of 1:1. A blank experiment was carried out byincubating 0.5 mL of the unactivated complex from Part D above (5×10⁻⁵mmol) with 1 mL of IgG solution (5×10⁻⁵ mol) for 2 hours.

G. Estimation of Conjugation Yirelds

Conjugation yields were estimated by gel permeation chromatography overSephadex™ G 75 column (30×1.4 cm). An aliquot of the reaction mixturewas loaded on the top of the column and eluted with 0.9% salinesolution. Two mL fractions were collected and the activity measured in aNaI (T1) scintillation counter. An equal amount of the sample loaded onthe column was diluted to 2 mL and kept as a control. Recovery from thecolumn was calculated by comparing the sum of the activity eluted fromthe column to the activity in the control tube. The conjugation yieldwas calculated by taking the ratio of the sum of the protein peak to thetotal recovered activity. The conjugation yield was about 80% at a 1:1ratio of complex to IgG.

When the unactivated complex was incubated with IgG and chromatographedin a Sephadex™ column, only 4% of the activity was eluted between 30 and50 mL, indicating no conjugation of the unactivated complex with theprotein.

The fraction with the maximum activity of the conjugated protein wassubsequently rechromatographed after 18 hours. Ninety percent of theactivity eluted with the protein on passing through a Sephadex™ G 75column, indicating that the conjugate was stable for that period oftime.

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of this specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A compound which is2,2,12,12-tetramethyl-4,7,10-triaza-7-(p-aminobenzyl)-1,13-tridecanedithiol.
 2. A complex which comprises a compound of the formula##STR13## wherein: R represents independently hydrogen, C₁ -C₃ alkyl, orbenzyl;R¹ represents --CH₂ C(CH₃)₂ SH, --(CH₂)₂ NH₂, --(CH₂)₂ SH,##STR14## Q represents hydrogen, C₁ -C₃ alkyl or phenyl; R² representshydrogen, --CH₂ CO₂ H, --CH₂ CH₂ CO₂ H, or --(CH₂)₂ NH₂ ; m and n areindependently 2, 3, or 4; L is a linker/spacer group covalently bondedto, and replaces one hydrogen atom of the nitrogen atom to which it isjoined, said linker/spacer group being represented by the formula##STR15## wherein: Y is selected from the group consisting of nitro,amino, isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl,bromoacetamido and maleimido; q is 1, 2, or 3; or a pharmaceuticallyacceptable salt thereof; and complexed with an ion of a metal selectedfrom the group consisting of ^(99m) Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ⁵⁷ Co, ¹⁸⁶ Re,¹⁸⁸ Re, ⁹⁷ Ru, ¹¹¹ In, ^(113m) In, ⁶⁷ Ga, and ⁶⁸ Ga.
 3. A complex ofclaim 2 wherein:m and n are 2 or 3; R² is hydrogen; R¹ is ##STR16## Y isamino or isothiocyanato; or a pharmaceutically acceptable salt thereof.4. A complex of claim 2 which is ¹⁰⁵ Rh withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine.
 5. A complexof claim 2 which is ^(99m) Tc withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine.
 6. A complexof claim 2 which is ⁵⁷ Co withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine.
 7. A complexof claim 2 which is ¹⁰⁵ Rh with1,7-bis(2-methylenepyrrole)-4-(p-aminobenzyl)diethylenetriamine.
 8. Acomplex of claim 2 which is ^(99m) Tc with2,2,12,12-tetramethyl-4,7,10-triaza-7-(p-aminobenzyl)-1,13-tridecanedithiol.
 9. A conjugate comprising a compound of the formula ##STR17##wherein: R represents independently hydrogen, C₁ -C₃ alkyl, or benzyl;R¹represents --CH₂ C(CH₃)₂ SH, --(CH₂)₂ NH₂, --(CH₂)₂ SH, ##STR18## Qrepresents hydrogen, C₁ -C₃ alkyl or phenyl; R² represents hydrogen,--CH₂ CO₂ H, --CH₂ CH₂ CO₂ H, or --(CH₂)₂ NH₂ ; m and n areindependently 2, 3, or 4; L is a linker/spacer group covalently bondedto, and replaces one hydrogen atom of the nitrogen atom to which it isjoined, said linker/spacer group being represented by the formula##STR19## wherein: Y is selected from the group consisting of nitro,amino, isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl,bromoacetamido and maleimido; q is 1, 2, or 3; or a pharmaceuticallyacceptable salt thereof;complexed with an ion of a metal selected fromthe group consisting of ^(99m) Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ⁵⁷ Co. ¹⁸⁶ Re, ¹⁸⁸Re, ⁹⁷ Ru, ¹¹¹ In, ^(113m) In, ⁶⁷ Ga, and ⁶⁸ Ga, with the proviso thatwhen R² is --CH₂ CO₂ H or --CH₂ CH₂ CO₂ H, then the radioactive metalion is selected from the group consisting of ¹⁸⁶ Re, ¹⁸⁸ Re, ^(99m) Tc,¹¹¹ In, ^(113m) In, ⁶⁷ Ga, and ⁶⁸ Ga; and covalently attached to aprotein or an antibody or antibody fragment.
 10. A conjugate of claim 9wherein:m and n are 2 or 3; R² is hydrogen; R¹ is ##STR20## Y is aminoor isothiocyanato, or a pharmaceutically acceptable salt thereof.
 11. Aconjugate of claim 9 which is the complex of ¹⁰⁵ Rh withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine to IgG.
 12. Aconjugate of claim 9 which is the complex of ^(99m) Tc withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine to IgG.
 13. Aconjugate of claim 9 which is the complex of ⁵⁷ Co withbis(2-hydroxybenzyl)-4-(-p-aminobenzyl)diethylenetriamine to IgG.
 14. Aconjugate of claim 9 which is the complex of ¹⁰⁵ Rh with1,7-bis(2-methylenepyrrole)-4-(p-aminobenzyl)diethylenetriamine to IgG.15. A conjugate of claim 9 which is the complex of ^(99m) Tc with2,2,12,12-tetramethyl-4,7,10-triaza-7-(p-aminobenzyl)-1,13-tridecanedithiol to IgG.
 16. A pharmaceutical formulation comprising a conjugatehaving a compound of the formula ##STR21## wherein: R representsindependently hydrogen, C₁ -C₃ alkyl, or benzyl;R¹ represents --CH₂C(CH₃)₂ SH, --(CH₂)₂ NH₂, --CH₂)₂ SH, ##STR22## Q represents hydrogen,C₁ -C₃ alkyl or phenyl; R² represents hydrogen, --CH₂ CO₂ H, --CH₂ CH₂CO₂ H, or --(CH₂)₂ NH₂ ; m and n are independently 2, 3, or 4; L is alinker/spacer group covalently bonded to, and replaces one hydrogen atomof the nitrogen atom to which it is joined, said linker/spacer groupbeing represented by the formula ##STR23## wherein: Y is selected fromthe group consisting of nitro, amino, isothiocyanato, semicarbazido,thiosemicarbazido, carboxyl, bromoacetamido and maleimido; q is 1, 2, or3; or a pharmaceutically acceptable salt thereof; andcomplexed with anion of a metal selected from the group consisting of ^(99m) Tc, ¹⁰⁵ Rh,¹⁰⁹ Pd, ⁵⁷ Co, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁹⁷ Ru, ¹¹¹ In, ^(113m) In, ⁶⁷ Ga, and ⁶⁸Ga, with the proviso that when R² is --CH₂ CO₂ H or --CH₂ CH₂ CO₂ H,then the radioactive metal ion is selected from the group consisting of¹⁸⁶ Re, ¹⁸⁸ Re, ^(99m) Tc, ¹¹¹ In, ^(113m) In, ⁶⁷ Ga, and ⁶⁸ Ga; andcovalently attached to a protein or an antibody or antibody fragment;and a physiologically acceptable carrier.
 17. A pharmaceuticalformulation of claim 16 wherein:m and n are 2 or 3; R² is hydrogen; R¹is ##STR24## Y is amino or isothiocyanato; or a pharmaceuticallyacceptable salt thereof.
 18. A formulation of claim 16 which is theconjugate of ¹⁰⁵ Rh withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine to IgG and apharmaceutically acceptable carrier.
 19. A formulation of claim 16 whichis the complex of ^(99m) Tc withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine to IgG and apharmaceutically acceptable carrier.
 20. A formulation of claim 16 whichis the complex of ⁵⁷ Co withbis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine to IgG and apharmaceutically acceptable carrier.
 21. A formulation of claim 16 whichis the complex of ¹⁰⁵ Rh with1,7-bis(2-methylenepyrrole)-4-(p-aminobenzyl)diethylenetriamine to IgGand a pharmaceutically acceptable carrier.
 22. A formulation of claim 16which is the complex of ^(99m) Tc with2,2,12,12-tetramethyl-4,7,10-triaza-7-(p-aminobenzyl)-1,13-tridecanedithiol to IgG and a pharmaceutically acceptable carrier.